IOS EDGE: Development of genetic tools for the dominant phototroph in the sea

浮游植物是微小的植物，构成了海洋食物链的基础，它们共同完成了地球上一半的光合作用，相当于陆地上所有的植物。原绿球藻是全球海洋中最丰富的单一浮游植物物种，每年产生约40亿吨固定碳，与全球农田的产量大致相同。此外，它们是自然界设计的最小和最有效的光合作用者，仅依靠阳光，二氧化碳和必需的营养物质来生长。在过去的25年里，对这种最小的原生生物的研究使人们越来越了解它在生物组织的各个尺度上的特征，从基因组到全球生态系统。这种迷人的微生物也为促进公众对海洋生态学、生物多样性和基因组学等主题的兴趣提供了一个很好的机会。它吸引了科学记者的注意，并充当了微生物的“大使”——例证了它们在地球维护中的重要作用。原绿球藻模型系统工具包中缺少的一件事是做遗传学的能力。迄今为止，这种细菌抵抗了所有基因操作的尝试，这极大地阻碍了发现的速度。该项目的目标是通过使用专门为该项目开发的传统和最先进的工具，开发一种有效的原绿球藻遗传系统，直接解决这一限制。在过去的25年里，随着越来越多的菌株被引入培养，原绿球藻的研究稳步扩大，它们在全球海洋中的分布已被广泛绘制。pi现在在实验室中有100个原绿球藻培养物，大量的转录组学和蛋白质组学数据，并且他们继续从培养物和野生种群中积累基因组数据。然而，每一个新的原绿球藻菌株，或单细胞测序，增加了大约100个全新的基因原绿球藻泛基因组，其中绝大多数是未知的功能。遗传系统的发展对于识别这些未知基因的功能和测试涉及具有假定功能的基因的假设是必要的。该项目特别涉及高通量微流体电穿孔技术，以比传统方法快几个数量级的速度转化顽固微生物。将对原绿球藻实施全局诱变（基于转座子的工具）和靶向诱变（基于crispr的工具）策略，同时优化微流体电穿孔技术，充分发挥其潜力。作为概念验证，每个工具都将通过使用我们目前已知的原绿球藻营养获取基因库来测试其有效揭示基因功能的能力。

Phytoplankton are microscopic plants that form the base of ocean food chains and collectively do half of the photosynthesis on Earth, equal to all the plants on land. Prochlorococcus is the single most abundant phytoplankton species in the global oceans and is responsible for producing an estimated 4 billion tons of fixed carbon each year, which is approximately the same productivity as global croplands. In addition they are the smallest and most efficient photosynthesizers designed by nature, relying only on sunlight, CO2 and essential nutrients for growth. For the past 25 years, research on this minimal prototroph has led to a growing understanding of its features across all scales of biological organization, from the genome to the global ecosystem. This fascinating microbe has also offered a great opportunity to foster public interest on topics such as ocean ecology, biodiversity and genomics. It attracts the attention of science journalists, and serves as an "ambassador" for microorganisms - exemplifying their important role in planetary maintenance. The one thing that is missing from the Prochlorococcus model system tool kit is the ability to do genetics. This bacterium has resisted all genetic manipulation attempts to date, which has significantly hampered the rate of discovery. The goal of this project is to directly address this limitation by developing an efficient genetic system for Prochlorococcus using both traditional and state-of-the-art tools developed specifically for this project.Research on Prochlorococcus has expanded steadily over the past 25 years, as more and more strains have been brought into culture, and their distribution in the global oceans has been mapped extensively. The PIs now have 100s of Prochlorococcus cultures in the lab, an extensive amount of transcriptomics and proteomics data, and they continue to accumulate genomic data from cultures and wild populations. However each new Prochlorococcus strain, or single cell sequenced, adds roughly 100 entirely new genes to the Prochlorococcus pangenome, the vast majority of which are of unknown function. The development of a genetic system is necessary for identifying the functions of these unknown genes and testing hypotheses involving those with putative functions. This project notably involves a high throughput microfluidic electroporation technology to transform recalcitrant microbes orders of magnitude faster than traditional approaches. A global mutagenesis (Transposon-based tools) and targeted mutagenesis (CRISPR-based tools) strategy will be implemented for Prochlorococcus, while optimizing the microfluidic electroporation technology to its full potential. As proof-of-concept, each tool will be tested in its ability to efficiently reveal gene functions by using our current known repertoire of nutrient acquisition genes in Prochlorococcus.